From|: Helene S. Barbosa
03/20/2011
To: Editor BMC Microcbiology
Letter to reviewers
MS: 1494086458504937
Toxoplasma gondii down modulates cadherin expression in skeletal muscle
cells inhibiting myogenesis
Alessandra Ferreira Gomes, Erick Vaz Guimarães, Laís Carvalho, José R.
Correa, Leila Mendonça-Lima and Helene Santos Barbosa
Reviewer number: 1
1. It is necessary to improve the literary quality of the manuscript.
Yes, the text was revised.
2. Several illustrations are not necessary since they show negative and/or
expected results where a mention to “not shown” could be used. Examples: (a)
Fig. 1, which show previously published observations, (b) Fig. 5, and (c) Fig. 9;
Ok, figures 1 and 5 were removed. In figure 9, only the graph was
maintained.
3. At the beginning of the Discussion section the authors list as a objective of
the present study “to analyse the T. gondii capacity to infect skeletal muscle
cells during myogenesis”. This is not a valid objective since it is well known that
T. gondii is able to infect all type of cells. Even the first descriptions of
cultivation in vitro of this protozoan used myoblasts;
Ok, the text was altered.
Lines: 287-291.
“This study analyzes the impact of T. gondii-infection on the myogenesis
process. The results obtained showed that: (i) myoblasts are more susceptible
to infection than myotubes; (ii) T. gondii-infected myoblasts are unable to fuse
1
with others myoblasts and myotubes and, (iii) M-cadherin expression is down
regulated during infection, indicating that T. gondii interferes with myogenesis in
SkMC model.”
4. Most of the discussion section is a repetition of the results and should be
considerably shortened. For instance phrases and paragraphs such as those
found in lines 323-325, 332-354, 366-371 are not necessary.
The text was revised and the cited phrases and paragraphs were
excluded.
Reviewer number: 2
Major compulsory revisions
1a. Fig 2. Clarification is necessary regarding how the percentage of infection
was determined. The materials and methods should contain a detailed
description of how the percentage of infection was determined, and the figure
legend should contain a brief explanation too.
Lines 119-124 in materials and methods were altered aiming to clarify the
determination of infected cells:
“T. gondii infection during skeletal muscle cell myogenesis “
Aiming to verify the infectivity of T. gondii in myoblasts and myotubes, we
developed the following protocol: 2-day-old cultures were infected with
tachyzoite forms (1:1 parasite-host cell ratio) and, after 24 h of interaction, the
total number of infected myoblasts and myotubes was quantified independent of
the number of internalized parasites. “
1b. Also, a different example should be shown for Fig 2B i.e., one that better
represents the data by showing a field of view with several infected myobasts
and fewer infected myotubes.
The figure 2B was excluded and changed by figure 1B and a new text in
results section was introduced:
Lines 219-224:
2
“Only the number of infected myoblasts and myotubes was evaluated,
independently of the number of parasites internalized. The total number of
infected cells (harboring at least one internalized parasite) after 24 h of SkMC parasite interaction represented 61% of myoblasts and 38% of myotubes. These
data indicate that myotubes were 1.6-fold less infected than myoblasts (Fig. 1A).
Figure 1B shows young and mature uninfected myotubes surrounded by several
heavily infected myoblasts after 48 h of interaction. “
Legend to figure 1 was changed: Lines 617-623
Fig. 1 - Percentage of T. gondii infected SkMC after 24 h of interaction. (A)
Percentage of myoblasts (61%) and myotubes (38%) infected with T. gondii
after 24 h of interaction. Student’s T-test (*) p ≤ 0.05. (B) Details of the profile of
SkMC cultures observed by fluorescence microscopy with phaloidin-TRITC
labeling showing actin filaments in red; nuclei of the cells and the parasites
labeled with DAPI, in blue. Infected cultures present myoblasts containing
several parasites (thick arrow) and young myotubes with 2 nuclei without
parasites (thin arrows). Bars, 20 μm.
2. Language needs correction throughout ….
The text was revised.
3. One conclusion from Fig 8 is that: “Myotubes infected reveal lower cadherin
distribution but they does not hinder the adhesion of uninfected myoblasts”. This
conclusion is unjustified without further evidence since it is not possible to
distinguish by microscopy whether two cells are adherent to one another. They
could have simply come in contact with one another due to migration or cell
division.
This conclusion was removed from the text.
Legend of figure 5 (old figure 8) was altered.
Lines 650-661:
Fig. 5 - Cadherin profile in differentiated cultures after more than 24 h of
interaction with T. gondii. (A and inset) Mature (arrowhead) and young
3
myotubes in fusion process with myoblasts (arrows) can be observed by phase
contrast microscopy. (B and inset) By fluorescence microscopy, cadherin (in
green) appears distributed throughout the myotubes, being more concentrated at
the cell membrane during adhesion, while mature myotubes alone show more
intense labeling at the extremities. (C) Interferential microscopy shows the
adhesion of uninfected myoblasts (arrowhead) with a mature infected myotube
(thick arrows). (D) Confocal microscopy analysis shows that the infected
myoblasts do not reveal cadherin labeling and more infected myotubes present
weaker cadherin labeling (arrow). Observe that despite the weak labeling, in
infected myotubes cadherin molecules appear to migrate to the point of contact
with uninfected myoblasts (arrowhead). (E) Merge. Bars, 20 μm
Minor compulsory revisions
1.
Fig. 1 legend title indicates the use of confocal but the figure only contains
DIC micrographs.
Yes. The fig. 1 was removed, as suggested by reviewer 1.
2. Fig 1. Zoomed-in insets would help the reader see the salient cellular
features without having the figure take up large amounts of print space.
Fig. 1 was removed, as suggested by reviewer 1.
3. Fig. 9D and E. It would be ideal to show an example where several parasites
are in the field of view, and also show the DIC image so that the reader can see
where the parasites are positioned.
Fig. 9 was removed, as suggested by reviewer 1.
4. It would be interesting to speculate in the discussion on what benefit it is to
the parasite to downregulate M-cadherin and inhibit myotube formation. Also,
do the findings have physiological relevance to T. gondii infection in vivo?
In this respect, the following texts were introduced in the discussion
section.
4
Lines 347-349:
“We believe that T. gondii, like other pathogens, can benefit from the
modulation of cadherin and other adhesion molecules in order to facilitate
migration to other neighboring cells and tissue.”
Lines 370-375:
“During embryonic development the formation and maintenance of muscle
tissues primarily requires the action of adhesion proteins such as cadherins
[43]. In our in vitro studies using SkMC we verified that T. gondii affected the
myogenesis process by negatively regulating cadherin expression. Thus, we
believe that our results can contribute to a further investigation of congenital
infection by Toxoplasma during the embryonic formation of muscle tissue.”
The following sections were also included in the manuscript:
Lines 404-412:
Authors’ contributions
HSB conceived, participated in the design and coordination of the study and
had the general supervision and complete overview of the project. AFG coconceived the study, carried out most of the experimental work, including the
processing of samples and the final illustrations for the manuscript, analyzed
data and drafted the manuscript, as part of her PhD thesis. EVG and LC
participated in the design of the study. JRC performed western blot analysis.
LML carried out the molecular assays. All authors analyzed the data and read
and approved the final manuscript.
Line 425: The authors declare that they have no competing interests.
5